Sterile sampling is a standard procedure in fermentation processes. It is the first operating step on the path to sample analysis for determining or detecting the state and quality of a bioprocess and, in particular, of the products arising from the latter. In this connection, it has hitherto been necessary, in many cases, for a laboratory technician to remove a sample manually. Delivery of the sample to a central analysis station is followed by sample preparation, i.e. biomass separation and aliquoting, and, finally, by the analysis on several different analysis devices. The analysis results are printed out for documentation of product quality and are input manually into databases and stored. Moreover, suitably identified reserve specimens are stored at low temperatures for subsequent detection methods. The analysis results are checked in the quality assurance system in order to release the product obtained in a bioprocess or to discard it because of quality defects. All of these steps are very labor-intensive and accordingly result in high costs. The process in the reactor is usually controlled and regulated after manual input of the analysis results obtained. Complete automation of process control and regulation is therefore not possible.
To reduce the number of staff needed, numerous automated individual components are now available on the market, e.g. sterile sampling devices, pipetting systems and automatic analyzers. However, complete automation of sampling and analysis is impossible because of the fact that samples have hitherto been transported exclusively by personnel from the production site to a separate laboratory for quality control, and because of the resulting break in the chain of automation. Moreover, laboratory analysis does not provide real-time information that would permit control of the process.
EP 1439472 A1 describes automatic process analysis, control and regulation, in particular by process chromatography, in chemistry and polymer science, where no particular demands are placed on sterility. The solution described does not satisfy the requirements of treating mechanically sensitive material, in particular biological material, and in particular living cells. The described process analysis system is unsuitable for most bioapplications.
Process chromatography for bioapplications is described in US 2004259241 A1 (Groton Biosystems). The described sampling device is limited to laboratory-scale bioreactors, since it is not sterilizable by steam, which is the usual sterilization method used in production. Moreover, Dionex Corporation offers the DX-800 process chromatograph (product brochure “DX-800 Process Analyser, Process Analytical Liquid Chromatography”) which can be operated for process control of bioapplications. This system offers automated chromatography, but no sampling that corresponds to the strict demands of sterilization technology on bioreactors. This system is additionally limited to the analysis of cell-free media. Both systems are designed for determination of several parameters. However, they do not provide an integrated solution for sampling, in particular of shear-sensitive material, e.g. cells, and no control and regulation of a bioprocess via the obtained data, since important automation units for connection to a process control system are lacking.
A further common disadvantage of the two systems described above for biotechnology applications is, in addition to there being no possibility of sterilization by steam, the fact that they are suitable only for sample preparation for a specific biochromatography process. Such systems cannot be used flexibly for other analysis methods, and instead are used exclusively for the particular biochromatography process described.
Sampling devices, and in particular sampling valves, for bioprocesses for removal of biological material and in particular cells are known as prior art, and some are even available commercially. For example, WO 1990012972 A1, from the company Keofitt a/s, describes a sampling valve composed of two parts, namely a valve body and a valve head. Inside the valve body, two connectors are connected via an annular channel about a rubber membrane. This structure allows the valve to be sterilized before and after use. WO 2004044119 A1 from Sartorius BBI Systems GmbH describes a coolable sampling valve with a cylindrical flow channel and a ram for blocking the flow channel, the walls of the sampling valve being made of a material of low thickness, usually of metal, in particular stainless steel, in order to permit rapid cooling of the valve, e.g. after steam sterilization. WO 1990012972 A1 and WO 2004044119 A1 do not describe the transport of the removed sample. A disadvantage of the two abovementioned sampling valves from WO 1990012972 A1 and WO 2004044119 A1 is that both have a metal-metal contact interface between the head of the sampling valve and the bioreactor, and, in the case of steam sterilization of the sampling valve, this causes local heating of the medium in the bioreactor, such that more cell aggregates may occur at the sampling site (so-called biofouling). When the described sampling valves are opened, such aggregates, which occur also during normal operation of a bioreactor, may swirl up and pass into the valve. To avoid clogging, transport conduits with relatively large diameters are therefore needed for transporting the removed cell suspension. In addition, the volume of the sample is determined exclusively by adjusting the opening time of the sampling valve. The solution does not permit precise removal of a predefined volume and in particular of a small volume. Moreover, no sample transport of a small cell-containing volume over a long distance is hitherto known without this sample, during transport, being adulterated, e.g. by sedimentation during transport or by destruction of cells by shearing.
There was therefore a need to make available an automatic and flexible process analysis system for control and regulation of bioprocesses with integrated connection to automation systems, which process analysis system permits sampling, gentle transport and preparation of small sample volumes containing mechanically sensitive material such as biological material, in particular living cells, and into which process analysis system a classical biochromatography system and/or further analyzers can be integrated. The process analysis system should be able to be operated under sterile conditions, and valves and transport conduits should be able to be sterilized as far as possible with steam, without causing heating of the reactor medium.